JP2002512208A - Improvements in or related to contrast agents - Google Patents

Abstract

  (57) [Summary] Volatile oils in water with gas-containing nucleation sites attached (eg, within droplets) to droplets of the dispersed oil phase to enhance the efficiency of the liquid-to-gas phase transition and control the phase transition The present invention provides a phase-change colloidal ultrasound contrast agent containing an emulsion having

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to ultrasonic imaging, in particular, a novel contrast preparation and ultrasonic imaging (
For example, its use in tissue perfusion visualization).

It is well known that contrast agents containing microbubble dispersions are particularly effective ultrasound backscattering agents due to the low density and ease of compression of the microbubbles. Such microbubble dispersions, if properly stabilized, are often very convenient, often at low doses, and are very effective, for example, for ultrasound visualization of microvasculature in the vascular system and tissues. Enable.

[0003] The use of sonography to assess blood perfusion (ie, blood flow per unit tissue mass) includes, for example, tumor detection (tumor tissue, which typically has a different vascular distribution than healthy tissue), and It has potential value in myocardial studies (eg, to detect myocardial infarction). A problem with the current use of ultrasound contrast agents for cardiac perfusion studies is that the information content of the resulting images is degraded due to the attenuation effect caused by the contrast agents present in the ventricles.

[0004] In our international patent joint application WO-A-9817324, the contents of which are incorporated herein by reference, which is incorporated herein by reference, we provide a controllable and temporary in- It has been disclosed that a gas-containing imaging formulation that promotes the growth of the gas phase in vitro can achieve and / or enhance the ultrasound visualization of perfusion in subjects, especially myocardium and other tissues. Such contrast agents may be used to promote controllable and transient gas phase retention, for example, in the form of microbubbles in tissue microvessels, thereby increasing gas concentrations in such tissues. Thus, echo generation (eg, associated with a blood pool) can be enhanced.

[0005] The use of gas as a tracer for such deposited perfusion differs significantly from current proposals for intravenously administrable microbubble ultrasound contrast agents. Therefore, it may generally be necessary to avoid the growth of microbubbles, which, if uncontrolled, can result in potentially dangerous tissue embolization. So limit the dose,
It may be necessary to use a gas mixture with the selected composition in order to minimize bubble growth in vitro by and / or to suppress internal diffusion of blood gas into the microbubbles. (See, for example, WO-A-9503835 and WO-A-9516467).

[0006] On the other hand, according to WO-A-9817324, a composition comprising a dispersed gas phase is a substance having a gas or vapor pressure sufficient to promote a controllable growth of the dispersed gas phase. ,
Or at least one substance capable of generating said gas or vapor pressure in vivo by internal diffusion of gas or vapor molecules derived from said substance (for the sake of simplicity, these substances are hereinafter referred to as "diffusible components" ), But it is understood that the effects of the present invention, in addition to or in place thereof, involve transport mechanisms other than diffusion, as described in more detail below. Will.

[0007] This simultaneous administration of a dispersed gas phase-containing composition and a composition containing a diffusible component with moderate volatility has been suggested by previous proposals for the administration of only volatile substances (eg WO-A- (In the form of phase shift colloids as described in 9416739). Thus, the contrast formulation of WO-A-9817324 allows for control of factors such as the probability and / or rate of growth of the dispersed gas by the selection of suitable components of the co-administered composition, Administration of the aforementioned phase change colloids alone may result in microbubbles that grow uncontrollably and irregularly, at least to the extent that they cause potentially dangerous embolization of the myocardial vasculature and brain, for example. (Eg, Schwarz, Advance in Echo-Contrast [1994 (3)], pp. 48-49).

[0008] Administration of phase change colloids alone may not cause reliable or consistent volatilization of the dispersed phase in vitro to generate gas or vapor microbubbles. Grayburn et al. (J. Am. Coll. Cardiol. 26 (5) [1995], pp. 1340-1347)
It has been suggested that pre-activation of the perfluoropentane emulsion may be required to achieve myocardial opacification in dogs at an effective imaging dose low enough to avoid hydrodynamic side effects. Techniques for activating such colloidal dispersions, including the application of low pressure thereto, are described in WO-A-9640282: this typically involves partially filling the emulsion into a syringe, followed by Forcibly pulling the plunger of the syringe and subsequently releasing it to produce a temporary pressure change that causes microbubbles to form in the emulsion. This is inherently a rather cumbersome technique and may not provide a consistent activation level.

With respect to phase change colloids, US-A-5536489 also discloses that an emulsion of a water-insoluble gas-forming chemical such as perfluoropentane may be used as a site-specific imaging contrast agent. The emulsions are described as subjecting ultrasound energy to specific locations in the body to be imaged only to generate significant amounts of image enhancing microbubbles. However, in our own studies, emulsions of volatile compounds such as 2-methylbutane or perfluoropentane, according to WO-A-9817324, were sufficient to give a significant contrast effect using a binary contrast agent. Ultrasound at energy levels has been shown to provide no detectable echo enhancement in vitro or in vivo.

WO-A-9725097 discloses the administration of an aqueous dispersion of superheated droplets of a water-immiscible liquid that can evaporate in vitro under the influence of radiation or ultrasound. It is said to induce uniform nucleation of the droplets. In particular, the dispersion can be used to form a diagnostic contrast agent, or to selectively deliver the agent to a limited area of the body.

The present invention is based on the observation that phase-change colloidal volatile emulsions in which gas-containing heterogeneous nucleation sites are attached to emulsion droplets have many valuable advantages. In particular, perfusion imaging can be performed in a manner similar to that described in WO-A-9817324, but without the need to administer two separate compositions, thereby facilitating product handling. In addition, factors such as the final size of the microbubbles generated by the volatile disperse phase, such as the size of the emulsion droplets and the nature and location of nucleation sites, can be easily set during the production of the contrast agent. It can be controlled by various parameters. Thus, the high-yield liquid-to-gas phase transition arising from the presence of nucleation sites allows for accurate prediction of the size of the microbubbles formed and allows for controlled retention with high safety. I do.

[0012] Thus, according to one aspect of the present invention, ultrasound imaging comprising an injectable oil-in-water emulsion wherein there is a gas-containing nucleation site associated with dispersed oil phase droplets. An agent is provided.

[0013] The present invention further provides an enhanced human or non-human animal subject comprising the step of injecting a contrast agent as described above into the vasculature of the subject to produce an ultrasound image of at least a portion of the subject. To provide a method for producing an image.

[0014] The dispersed oil phase contains one or more suitable volatile components, at least one component being at least partially water-insoluble or water-immiscible. The component or mixture of components is advantageously liquid at the production and storage temperatures (eg, may be as low as −10 ° C. if the aqueous phase contains a suitable cryoprotectant),
On the other hand, at body temperature it is gaseous or exhibits a sufficient vapor pressure (at least 50
mmHg, preferably at least 100 mmHg). Also, if desired, other substantially water-insoluble and water-immiscible components having lower volatility may be present in the oil phase.

Suitable volatile components are, for example, the emulsifiable low-boiling liquids provided in WO-A-9416739 mentioned above, the contents of which are incorporated herein by reference. May be selected from various lists. Specific examples of emulsifiable oil phase components include:
Aliphatic ethers such as diethyl ether; polycyclic oils or alcohols such as menthol, camphor or eucalyptol; heterocyclic compounds such as furan or dioxane; n-butane, n-pentane, 2-methylpropane; 2-
Methylbutane, 2,2-dimethylpropane, 2,2-dimethylbutane, 2,3-
Dimethylbutane, 1-butene, 2-butene, 2-methylpropene, 1,2-butadiene, 1,3-butadiene, 2-methyl-1-butene, 2-methyl-2-butene, isoprene, 1-pentene, Aliphatic hydrocarbons (saturated or unsaturated, and linear or branched) such as 1,3-pentadiene, 1,4-pentadiene, butenin, 1-butyne, 2-butyne or 1,3-butadiyne; cyclobutane, cyclobutene And cycloaliphatic hydrocarbons such as methylcyclopropane or cyclopentane; and low molecular weight halogenated hydrocarbons (eg, containing up to 7 carbon atoms).
Representative halogenated hydrocarbons are dichloromethane, methyl bromide, 1,2-dichloroethylene, 1,1-dichloroethane, 1-bromoethylene, 1-chloroethylene, ethyl bromide, ethyl chloride, 1-chloropropene, 3 -Chloropropene, 1-
Includes chloropropane, 2-chloropropane and t-butyl chloride. Preferably,
Some of the halogen atoms are fluorine atoms, for example, dichlorofluoromethane,
Trichlorofluoromethane, 1,2-dichloro-1,2-difluoroethane, 1
, 2-Dichloro-1,1,2,2-tetrafluoroethane, 1,1,2-trichloro-1,2,2-trifluoroethane, 2-bromo-2-chloro-1,1,1
-Trifluoroethane, 2-chloro-1,1,2-trifluoroethyl difluoromethyl ether, 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether, partially fluorinated alkanes (e.g., 1H Pentafluoropropane, such as, 1H, 3H-pentafluoropropane, hexafluorobutane, nonafluorobutane, such as 2H-nonafluoro-t-butane, and 2H, 3
Decafluoropentane, such as H-decafluoropentane), partially fluorinated alkenes (eg, heptafluoropentene, such as 1H, 1H, 2H-heptafluoropent-1-ene, and 1H, 1H, 2H-). Nonafluorohexa
Nonafluorohexenes such as 1-ene), fluorinated ethers (e.g., 2
, 2,3,3,3-pentafluoropropyl methyl ether, or 2,2,3
3,3-pentafluoropropyldifluoromethyl ether), and more preferably perfluorocarbon. Examples of perfluorocarbons include perfluorobutane, perfluoropentane, perfluorohexane (eg, perfluoro-2-methylpentane), perfluoroheptane, perfluorooctane,
Perfluoroalkanes such as perfluorononane and perfluorodecane; perfluorocycloalkanes such as perfluorocyclobutane, perfluorodimethyl-cyclobutane, perfluorocyclopentane and perfluoromethylcyclopentane; perfluorobutene (e.g. perfluorobut-2-ene or perfluorobuta-1) , 3-diene), perfluoropentene (e.g., perfluoropent-1-ene) and perfluorohexene (e.g., perfluoro-2-methylpent-2-ene or perfluoro-4-methylpenta-2)
Perfluoroalkenes such as -ene); perfluorocycloalkenes such as perfluorocyclopentene or perfluorocyclopentadiene; and perfluorinated alcohols such as perfluoro-t-butanol.

Such at least partially water-insoluble / water-immiscible volatiles may include soluble substances that greatly increase the vapor pressure of the mixture. Such solutes include air; nitrogen; oxygen; carbon dioxide; hydrogen; an inert gas such as helium, argon, xenon or krypton; sulfur hexafluoride, disulfur defluoride or trifluoromethyl pentafluoride. Sulfur fluorides such as sulfur; selenium hexafluoride; optionally halogenated silanes such as methylsilane or dimethylsilane; methane;
Alkanes such as ethane, propane, butane or pentane; cycloalkanes such as cyclopropane, cyclobutane or cyclopentane; alkenes such as ethylene, propene, propadiene or butene; or alkynes such as alkynes such as acetylene or propyne. Low molecular weight hydrocarbons (eg, containing up to 7 carbon atoms); ethers such as dimethyl ether; ketones; esters; low molecular weight halogenated hydrocarbons (eg, containing up to 7 carbon atoms); or any of the foregoing. Gases such as mixtures are included. More preferred are gases such as air, oxygen and carbon dioxide that are substantially soluble in the fluorocarbon liquid.

[0017] The emulsion will typically be stabilized by one or more surfactants or other encapsulating substances. It will be appreciated that the nature of such materials can greatly affect factors such as the rate of growth of the vaporized gas. Generally, a wide range of surfactants will be used, selected from the extensive list provided, for example, in EP-A-0727225, the contents of which are incorporated herein by reference. Representative examples of useful surfactants include fatty acids (eg, straight-chain saturated or unsaturated fatty acids containing 10-20 carbon atoms) and their sugar esters and triglyceride esters, phospholipids (eg, lecithin or fluorine-containing Phospholipids), proteins (eg, albumin such as human serum albumin), block copolymer surfactants (eg, polyoxyethylene-polyoxypropylene block copolymers such as Pluronics, or acyloxyacyl polyethylene) Glycol (for example, polyethylene glycol methyl ether 16-
Extended polymers such as hexadecanoyloxyhexadecanoate, where, for example, the polyethylene glycol moiety has a molecular weight of 2300, 5000 or 10,000), fluorine-containing surfactants (eg, sold under the trade names Zonyl and Fluorad) Or described in WO-A-9639197, the contents of which are incorporated herein by reference, and one or more quaternary ammonium groups and long-chain alkyls ( For example, cationic surfactants containing one or more lipid or alkanoyl groups such as C _10-30 ) are included.

The emulsion droplets may also be incorporated by a wall-forming encapsulant because the dispersed phase is in the form of microcapsules containing a volatile liquid, or in a porous structure such as latex particulates. May be stabilized. Representative wall-forming substances include polylactic acid, polycaprolactone, polycyanoacrylate and polyester (as described, for example, in WO-A-9317718).

The nucleation site may be present in the dispersed oil phase droplets or in a surfactant or other encapsulating or stabilizing film surrounding the droplets; such a film Function as nucleation sites on their own. Alternatively, a suitable nucleation site may be in contact with the outside of such a membrane.

When the nucleation sites are present in oil droplets, they are, for example, free microbubbles,
Surfactants or lipid stabilized microbubbles, polymer or protein encapsulated microbubbles, gas-containing porous solid microparticles such as aerogels or zeolites, trapped in pores or other irregularities of coarse surface solid microparticles Gas, gas-containing polymer microparticles, or fullerenes, clathra
It may take the form of dispersed microbubbles, such as gaseous inclusions such as tes) or nanotubes. Such contrast agents are quickly prepared by dispersing the nucleation site-containing material in the oil phase using one or more suitable dispersants, followed by forming an oil-in-water emulsion in a manner known per se. It is possible.

For easy dispersion, the interfacial properties of the nucleation site may be determined, for example, by selection of a dispersant for the nucleation site, or by chemical modification of the nucleation site surface (eg, silanisation).
Or by plasma modification). Also, the presence of surface irregularities, cavities, boundaries, gaps, or other structural defects is advantageous because it helps the gaseous phase spread to the interface.

If desired, the nucleation sites may be selected to have interfacial properties such that they can be located at the water-volatile oil interface. This can be achieved, for example, by choosing a dispersant for the nucleation site such that the surface of the nucleation site can be partially wetted by both the volatile oil and the aqueous phase. If necessary, the surface of the nucleation site may be adjusted by chemical modification (eg, plasma modification), rinsing, etc.

In an embodiment of the present invention, if it is desired that the generation of microbubbles occur spontaneously in vivo, the boiling point of the dispersed oil phase of the emulsion should not exceed 42 ° C., ie, from the volatile components of the oil phase. Is generally preferred to exceed 1 atmosphere at 42 ° C.

In another embodiment of the present invention, the microbubbles are formed by suitable temperature and / or pressure modification or application of an external activating force such as sound, ultrasound or radiation.
It may occur in vivo or immediately prior to injection. Also, when an external activating force is applied, an emulsion in which the oil phase has a higher boiling point (eg, up to 60 ° C.) may have an external activation despite the boiling point being farther away from the body temperature. May be useful because the oil phase will be largely evaporated in vivo.

The microbubbles generated from the contrast agents according to the present invention are characterized by an easily controllable growth rate and final size; they are controlled, for example, in tissue microvessels (eg myocardium). It may be designed to grow to a size of, for example, 10-20 μm, so as to exhibit a controlled retention, and they may be free-flowing.
It may be designed to grow to a size of, for example, 1-7 μm to act as a contrast agent.

In the presence of nucleation sites, the liquid-to-gas phase change in the emulsion droplets ensures a very efficient and rapid conversion of the liquid and thus the diffusion of volatiles between the individual particles It will be appreciated that this limits the air flow and thus the uncontrolled bubble growth. In this regard, the material inside one emulsion droplet can be converted into one foam. Assuming a gas represented by the ideal gas law [Equation (1)], pV = nRT (1) where n is the number of moles of a substance for creating one bubble, and the radius of the emulsion droplets, associated with r _e,

(Equation 1)

Here, if d is the density of the liquid phase, M _w is the molecular weight of the volatile substance, and V _e is the volume of the liquid droplet, the equation (1) of the ideal gas law can be obtained. put equation (2), when the volume V of the resulting foam is expressed by radius r _b, the following equation is obtained.

[0028]

(Equation 2)

For a typical volatile solvent, for example perfluoropentane, d is 1.66 g
_{/ Ml, M w = 288g /} mol and T = 298K, the use of p = 1 atm, r _b ≒ 5.2r _e. Thus, to provide microbubbles of size 10 μm that can be temporarily retained, the emulsion droplets have a size just below 2 μm.

For those nucleation sites that occupy 50% of such emulsion droplets, their size should be less than 1.6 μm. More preferably, the nucleation sites should occupy less than 20% of the emulsion droplets, and therefore have a size of 1.
Should be less than 2 μm; more preferably the nucleation site is 10% of the liquid volume
And should have a size of less than 1 μm.

To ensure that a sufficient number of emulsion droplets evaporate, a sufficient number of nucleation sites should be added. The nucleation sites are distributed on the liquid carrier particles by a simple Boltzmann distribution to estimate the amount of nucleation sites added for a certain percentage of the liquid carrier particles to contain at least one nucleation site. May be calculated.

The liquid to gas phase transition may be activated by simply heating to a temperature above the boiling point of the volatile liquid. Volatile oils with boiling points below body temperature should be used to activate the phase transition upon injection, taking advantage of the fact that the temperature rises to body temperature. However, also at higher temperatures, volatile liquids may have a boiling point well below body temperature, as the rate of bubble nucleation may be low. In such superheated dispersions, the presence of nucleation sites can lower the barrier to phase change so that nucleation can be caused by external influences.

Products whose gas formation is activated by treatment such as ultrasound are particularly advantageous in that they can be very storage-stable before activation and before use.

[0034] The dispersed gaseous content of the contrast agent according to the present invention will tend to be temporarily retained in the tissue at a concentration proportional to the local rate of tissue perfusion. Thus, using conventional imaging, or an ultrasound imaging modality such as harmonic B-mode imaging, where the display is derived directly from the reflected signal intensity, such tissue images are such that the displayed signal intensity is a function of local perfusion. It can be interpreted as a perfusion map. This is in contrast to images obtained with a free-flowing contrast agent where the local concentration of the contrast agent and the corresponding reflected signal intensity depend on the actual blood volume rather than the local tissue perfusion rate.

In studying the heart when a perfusion map is obtained from the reflected signal intensities according to this aspect of the invention, the differences, and therefore, between any myocardium regions supplied by the normally perfused myocardium and the stenotic artery, It may be advantageous to subject the patient to physical or pharmacological stress to enhance the difference in image intensity at the patient.
As is known in radionuclide heart imaging, such stress causes vasodilation and increased blood flow in healthy myocardial tissue, whereas blood flow in hypoperfused tissue supplied by stenotic arteries is It does not change substantially because the vasodilator capacity of the small arteries has already been exhausted by the natural self-regulation to increase the restricted blood flow.

The application of stress, such as physical exercise or the administration of pharmacologically adrenergic agonists, causes discomfort, such as chest pain, in a group of patients potentially with heart disease, Adenosine, dipyridamole, nitroglycerin, isosorbide mononitrate, prazosin, doxazosin, dihydralazine, hydralazine, sodium nitroprusside, pentoxyphylline
line), amerodipine (amelodipine), felodipine, isradipine (isradipine)
Preferably, perfusion of healthy tissue is increased by administration of a vasodilator such as selected from nifedipine, nimodipine, verapamil, diltiazem and nitrous oxide. In the case of adenosine, this results in a more than 4-fold increase in the coronary blood flow of healthy myocardial tissue, greatly increasing the uptake and temporary retention of the contrast agent according to the invention, and thus of normal and hypoperfused myocardial tissue. Greatly enhance the difference in reflected signal strength between the two. Due to the complexity of the physical entrapment process, retention of the contrast agent according to the invention is very effective; this is because radionuclide tracers such as thallium-201 and technetium sestamibi. (This is limited by the short contact time between the tracer and the tissue, and may therefore require maintenance of vasodilation during the entire distribution of the tracer's blood pool (eg, thallium scintigraphy) 4-6 minutes))). On the other hand, the contrast agents of the present invention do not suffer from such diffusion and transport restrictions and, due to the above-mentioned method, can quickly terminate their retention in myocardial tissue, and therefore have the The duration of vasodilation required to achieve cardiac perfusion imaging according to aspects can be very short (eg, less than 1 minute). This will reduce the duration of any possible discomfort to the patient due to vasodilator administration.

Adenosine is a particularly useful vasodilator in view of the fact that the required vasodilation only needs to be kept short, which is an endogenous substance and the half-life of the blood pool is only a few seconds As can be seen, it has a very short lasting effect. Thus, vasodilation will be strongest in the heart because the drug tends to reach distant tissues in lower than pharmacologically effective concentrations. Due to this short half-life, repeated injections or infusions of adenosine may be required during cardiac imaging according to this aspect of the present invention; Substantially at the same time as the administration of the agent composition, followed by an additional slow injection of adenosine 150 μg / kg (eg, over 20 seconds) 10 seconds later.

The contrast agent of the present invention can be usefully used for therapeutic applications such as drug delivery agents. Thus, the hydrophobic drug may be dissolved in the volatile oil phase to advantageously achieve a high drug load. Also, the treatment may be incorporated into any encapsulating membrane, or may be dissolved in the aqueous carrier phase. The treatment may also be present as nano- or micro-sized particles that serve as additional nucleation sites.

Without being bound by theoretical considerations, evaporation of the volatile oil droplets increases the drug concentration in the liquid droplets and easily exceeds solubility, thus speeding the release of the dissolved therapeutic agent. Is believed to be. Drug uptake is also increased by effects from "microstreaming" induced by local shifts and microbubble formation.

According to yet another aspect of the invention, the induced liquid to gas transition may be utilized for applications such as ultrasound therapy. Thus, for example, after the appropriate application of ultrasonic waves locally, the phase transition from liquid to gas provides microbubbles that are large enough to form an embolus in the capillary, and thus the site of interest (eg, , Tumors) can be prevented. Also, the microbubbles can absorb ultrasonic energy, and thus can heat the site of interest and are used for high heat treatment. In addition, the transition from liquid to gas is very rapid and can provide shear stress or microcurrent with a loss effect on surrounding cells; this can be useful for cell killing, eg, cancer treatment.

The following non-limiting examples illustrate the invention.

Example 1 Kaolin having a fine spatula edge was used as perfluoropentane containing 0.2 ml of a Fluorad® FC-171 surfactant (boiling point: 28 ° C.)
) Add to 2 ml. After gentle hand stirring, a milky white dispersion is obtained. 0.1 ml of the above dispersion and 1 ml of water are mixed by stirring with Espe Capmix (registered trademark) for 30 seconds to obtain an emulsion having droplets having a size slightly exceeding 1 μm.

The microscopic operation is performed while placing the emulsion droplets on a cooling / heating table and heating to 37 ° C. Several 10 μm droplets were observed, demonstrating a rapid liquid-to-gas phase change in the emulsion droplets.

The tube containing the emulsion is immersed in a water bath maintained at 37 ° C. and only a portion of the emulsion is heated. The turbidity of the heated emulsion is greatly increased immediately compared to the unheated emulsion, demonstrating the formation of small bubbles after heating.

Example 2 A zeolite having a fine spatula edge was prepared by adding perfluorooctanoic acid to 0.1 μg perfluorooctanoic acid.
Add to 2 ml of perfluoropentane (boiling point 28 ° C.) containing 2 mg. While keeping the sample in an ice bath, a Branson W385 sonicator horn (Branson W385 so
The sample is sonicated for 2 minutes at 50% power using a nicator horn).
0.1 m of the above dispersion and 1 ml of water are mixed by stirring for 45 seconds using Espe Capmix (registered trademark) to obtain an emulsion.

A sample (1 μl) of the emulsion was taken at room temperature with Isoton II (55 ml).
1) and measure the attenuation of the sound wave as a function of time at a center frequency of 3.5 MHz and 5.0 MHz, respectively, using two broadband transducers in a pulse-echo technique. The sound wave is weakly attenuated. Subsequently, the sample is heated step by step,
Measure the sound attenuation at each temperature. When the sample temperature is around 30 ° C., a substantial increase in sound wave attenuation is observed. This experiment illustrates how a nucleation site-containing emulsion with volatiles is converted to a microbubble dispersion near its boiling point. It also demonstrates the change in acoustic properties and the usefulness of the product as an ultrasound contrast agent.

Example 3 (Comparative Example) Example 2 is repeated without adding the finely divided zeolite to the perfluoropentane phase. Characterizing the emulsion using acoustic attenuation measurements techniques, heating to temperatures well above 40 ° C. results in only a slight increase in acoustic attenuation. This indicates the need for a nucleation site attached to the dispersed phase.

Example 4 a) 5 ml of a 5% W / V solution of the polymer obtained from Example 2 (a) of WO-A-9607434 in (−)-camphene at 60 ° C., and human serum albumin in water At the same temperature to 15 ml of a 5% W / V solution of The mixture is mixed at 20,000 rpm with an Ultra Turax T25 mixer at 1 20,000 rpm.
Mix vigorously for minutes. The sample is then passed five times using an Emulsiflex C5 high pressure homogenizer at a maximum pressure of 200,000 kpa and the emulsion is homogenized at 60 ° C. The median size of the obtained emulsion is around 300 nm. Subsequently, the emulsion is frozen in a dry ice / methanol bath and lyophilized for 48 hours to obtain a white powder. Electron microscopy shows the formation of gas-filled nanocapsules. The polymer particles are dispersed in water and excess human serum albumin is removed by dialysis.
The remaining polymer nanocapsules are dried under reduced pressure.

B) Add spatula-edge nanocapsules stabilized with the washed hollow polymer obtained from (a) above to 2 ml of perfluorodimethylcyclobutane (boiling point 45 ° C.) containing 0.2 ml of perfluorooctanoic acid I do. The sample is stirred for 1 hour with a laboratory shaker, resulting in a dispersion of nanocapsules filled with gas dispersed in perfluorodimethylcyclobutane. 0.1 ml of the above dispersion and 1 ml of water are mixed by stirring for 45 seconds using Espe Capmix (registered trademark) to obtain an emulsion.

C) placing the droplets of the emulsion obtained from (b) above on a cooling / heating table,
The microscope is operated while heating to 50 ° C. When the temperature exceeds 45 ° C, 1
Several 0 μm droplets were observed, demonstrating a rapid liquid to gas phase change in the emulsion droplets.

D) The tube containing the diluted emulsion obtained from (b) above is immersed in a water bath maintained at 50 ° C., and only a part of the emulsion is heated. In the heated part of the emulsion, the turbidity is immediately greatly increased compared to the unheated emulsion, demonstrating the formation of small bubbles on heating.

E) A sample (1 μl) of the emulsion obtained from (b) above was suspended in Isoton II (55 ml) at room temperature and pulsed echo technique using two broadband transducers, respectively. Center frequency 3.5MHz and 5.0MH
At z, the attenuation of the sound wave is measured as a function of time. The sound wave is weakly attenuated. Subsequently, the sample is heated stepwise, and the attenuation of the sound wave is measured at each temperature. When the sample temperature is between 35 and 40 ° C., a substantial increase in sound attenuation is observed. This experiment is
It illustrates how an emulsion containing nucleation sites with volatiles is converted to a microbubble dispersion well below its boiling point when the emulsion is exposed to external ultrasound. It also demonstrates changes in the properties of sound waves and the usefulness of the product as an ultrasound contrast agent.

Example 5 A dog is anesthetized, a median sternotomy is performed, and the anterior pericardium is removed. Using the ATL HDI-3000 scanner equipped with a P3-2 transducer, midline short axis B mode imaging of the heart is performed through a low attenuation 30 mm silicone rubber spacer. The frame rate is 40 Hz and the mechanical index is 1.1. 0.2μ
Example 4 of an amount corresponding to 1 (perfluorodimethylcyclobutane) / kg (body weight)
The perfluorodimethylcyclobutane emulsion containing polymer nanocapsules of b) is injected intravenously into dogs. There is a substantial increase in echo intensity from the myocardium, which begins approximately 20 seconds after injection and lasts 20 minutes. When the contrast agent is almost empty in the ventricles, there is an increase in myocardial opacity. Therefore, the observed effect is
Due to microbubbles delayed in the myocardium.

Example 6 (Comparative Example) Example 5 is repeated except that the polymer nanocapsules are not used and the perfluorodimethylcyclobutane emulsion phase is used. In vitro ultrasound imaging shows the limited sonic effect of emulsions. This comparative experiment shows that a nucleation site filled with gas bound to the emulsion droplets is required.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] August 3, 2000 (2008.3.3)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 1

[Correction method] Change

[Correction contents]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/14 A61K 47/14 47/24 47/24 47/34 47/34 47/42 47/42 ( 81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW) , EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN , CU, CZ, D E, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventors Skurtbait, Roald, Norway, N-0401 Oslo, Postbox 4220 Niidalen, Niikobeien 1-2, Nicomd Within Imaging AS (72) Inventors Wiggen, Unni Nordby Norway, N-4001 Oslo, Postbox 4220 Niidalen, Niikobeien 1-2, Nicomed Imaging S (72) Inventor Ostensen, Johnny Norway, N-4001 Oslo, Postbox 4220 Niidalen, Niikobeen 1-2, Niikom Imaging IFS F-term (reference) 4C076 AA17 BB12 CC50 DD34 DD35 DD37 DD39 DD41 DD44 DD58 DD60 DD63 EE41 EE49 FF70 4C085 AA40 HH09 JJ03 KA16 KA36 KB39 KB42 KB56 KB60 KB67 KB82 LL01 LL07

Claims (22)

[Claims] 1. An ultrasound contrast agent comprising an injectable oil-in-water emulsion, wherein the gas-containing nucleation site is associated with dispersed oil phase droplets. 2. The method according to claim 1, wherein said nucleation sites are present in dispersed oil phase droplets.
The contrast agent according to item 1. 3. The contrast agent according to claim 2, wherein the nucleation site is a free microbubble, a microbubble stabilized with a surfactant or a lipid, a microbubble encapsulating a polymer or a protein, and containing a gas. A contrast agent containing porous solid fine particles, gas-containing rough surface solid fine particles, gas-containing polymer fine particles, or gas-containing fullerenes, clathrates or nanotubes. 4. The contrast agent according to claim 1, wherein the nucleation site is present in a film that stabilizes the dispersed oil phase droplets or is in contact with the outside of such a film. 5. The oil phase according to claim 1, wherein the oil phase is at least one component selected from aliphatic ethers, polycyclic oils and alcohols, heterocyclic compounds, aliphatic hydrocarbons, cycloaliphatic hydrocarbons and halogenated hydrocarbons. 5. The contrast agent according to claim 1, wherein the component has a boiling point not exceeding 60 ° C. 6. 6. The contrast agent according to claim 5, wherein the oil phase contains one or more perfluorocarbons. 7. The contrast agent according to claim 6, wherein the perfluorocarbon is perfluorobutane, perfluoropentane, perfluorohexane, perfluorocyclobutane, perfluorodimethylcyclobutane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorobutene, Perfluorobutadiene, perfluoropentene, perfluorohexene, perfluorocyclopentene, perfluorocyclopentadiene and perfluoro-t-
A contrast agent selected from butanol. 8. The contrast agent according to claim 1, wherein the oil phase contains a gas solute. 9. The contrast agent according to claim 8, wherein the oil phase contains air, oxygen or carbon dioxide dissolved in a liquid fluorocarbon. 10. The contrast agent according to any one of claims 1 to 9, wherein the dispersed oil phase droplets are composed of a fatty acid, a sugar ester and a triglyceride ester of a fatty acid, a phospholipid, a protein, and a block. A contrast agent which is stabilized by a surfactant selected from a copolymer surfactant, a fluorine-containing surfactant and a cationic surfactant. 11. The method of claim 1, wherein the dispersed oil phase droplets are stabilized by a polymer wall forming encapsulating material or by incorporation into porous latex microparticles. Contrast agent. 12. The oily phase having a boiling point not exceeding 42 ° C.
The contrast agent according to any one of the above. 13. A composite preparation for simultaneous use, separate use or continuous use as a contrast agent in ultrasound imaging, wherein (i) the contrast agent according to any one of claims 1 to 12, And (ii) a preparation containing a vasodilator. 14. The combination preparation according to claim 13, wherein the vasodilator is adenosine. 15. A drug delivery agent containing the contrast agent according to any one of claims 1 to 12 together with a therapeutic agent. 16. The drug delivery agent according to claim 15, wherein a hydrophobic drug is dissolved in the oil phase. 17. The drug delivery agent according to claim 15, wherein the therapeutic agent is present as nano-sized or micro-sized particles. 18. A method for producing an enhanced image of a human or non-human animal subject, comprising injecting the vasculature of the subject with the contrast agent of any one of claims 1-14. Producing an ultrasound image of at least a portion of said subject. 19. The method of claim 18, wherein the growth of microbubbles from the contrast agent is activated in the subject by applying an external activation. 20. The method of claim 19, wherein said external activation comprises ultrasonic irradiation. 21. Use of a contrast agent according to any one of claims 1 to 12 in ultrasound therapy. 22. The use according to claim 21, wherein said treatment comprises cell killing or blocking blood flow to a site of interest.